1. Field of the Invention
The present invention relates to inhibitors of Staphylococcus SarA protein function involved in the expression of staphylococcal virulence factors and the use of these inhibitors to treat and prevent staphylococcal infections in subjects. Particularly, the inhibitors act to interfere with the binding of the SarA protein to its binding site(s). The selection of specific inhibitors of the SarA protein is made possible as a result of the identification of the binding sites of SarA protein on at least a portion of the agr (accessory gene regultor) gene, a gene that like the sar (staphylococcal accessory regulator) gene, plays a role in the virulence of Staphylococcus.
There is a great and urgent need among infectious-disease specialists, who have begun seeing one of their worst nightmares come true. They may be losing their last line of defense against the dangerous pathogen Staphylococcus aureus (S. aureus), which causes infections ranging from skin abscesses to such life-threatening conditions as pneumonia, endocarditis, septicemia, and toxic shock syndrome. Roughly one-third of the strains currently isolated from patients who acquire S. aureus infections while hospitalized are resistant to all antibiotics but one, vancomycin and now resistance to that antibiotic is cropping up. The present invention provides a new approach to combating S. aureus that may sidestep the organism""s ability to develop resistance.
Despite intensive research efforts over the past 50 years, Staphylococcus, particularly Staphylococcus aureus, remains a serious threat to human health. In fact, recent reports describe clinical isolates with reduced susceptibility to vancomycin. Therefore, S. aureus represents a bigger threat to human health now, than at any time since the pre-antibiotic era.
Staphylococcus is an opportunisitic bacteria that takes advantage of immunocompromised subjects and may become pathogenic in these subjects. There are approximately thirty-two species of Staphylococcus with only three consistently causing human disease. S. aureus is clearly the most prominent disease causing species, followed by S. epidermidis, and in a distant third is S. saprophyticus. S. epidermidis is becoming more prominent as a disease causing species because it causes infections of in-dwelling medical devices. As a result, researchers are looking more carefully at S. epidermidis, and as a result of this research, have found homologs of both the sar and agr genes in S. epidermidis. Fluckiger, U., et al. (1998). Otto,M., et al. (1998), respectively.
S. aureus can cause a diverse array of diseases ranging from relatively superficial infections of the skin (boils) to infections of the eye (endopthalmitis) to life threatening osteomyelitis, endocarditis and toxic shock syndrome (reviewed by Projan and Novick, 1997). S. aureus is armed with a large battery of virulence factors that enable it to colonize a human host and cause a variety of disease states (reviewed by Projan and Novick, 1997). Nosocomial infections are of particular concern for two reasons. The first is that the majority of life-threatening infections arise in the hospital environment. For example, while the frequency of S. aureus infections incurred during orthopedic or cardiac implant surgery is steady, the overall number of infections has risen dramatically in the past decade. This is largely due to the increase in the frequency of these procedures. S. aureus has an amazing capacity to colonize in-dwelling prosthetic devices. The second reason for increased concern of S. aureus infections is that strains of methicillin-resistant Staphylococcus aureus (MRSA) are endemic in hospitals. Moreover, strains with some resistance to vancomycin emerged in the United States in 1997 (Tenover et al., 1998; Smith et al., 1999; Sieradzki et al., 1999). Therefore, the need for new, effective treatments for this drug resistant pathogen is urgent.
The variety of virulence factors expressed by S. aureus contribute to a highly efficient system for survival. Early in the infection, surface proteins are predominantly expressed. Protein A and the adhesins (e.g., collagen, fibronectin) are representative surface proteins that solve two problems for the S. aureus cell. First, they bind to extracellular matrix components and anchor the cell to the host tissue. Second, they provide a host protein camouflage which helps the infecting cell elude the host""s immune system. The nascent colony increases in size until a critical number of cells is achieved (quorum) and a switch is thrown to re-organize the expression of virulence factors from surface proteins to exoproteins. These latter factors contribute to sequestration of the colony within a protective biofilm and enzymatic degradation of host tissue with an army of digestive enzymes, such as nucleases, lipases and proteases, which eventually result in an abscess. These enzymes accomplish two important functions or the bacterium: (1) allowing space for growth of the colony by getting rid of host tissue and (2) digested host tissue is assimilated by the bacterial cells for growth. Deep-seated abscesses, such as those found in staphylococcal ostemyelitis and endocarditis, often require surgical intervention to remediate the disease. It is important to note that this phenotypic switching process can be largely recapitulated in the laboratory environment, with surface protein expression occurring in the early log phase of a culture""s growth and exoprotein expression occurring late in log and into the stationary phases of growth.
The potency of this pathogen can be attributed to the coordinated, temporally-regulated expression of a wide array of virulence factors. Early in infection expression of surface proteins predominates, e.g., the collagen and fibronectin adhesins and protein A. The surface proteins allow the organism to attach to host tissues and evade the immune system. However, when the concentration of S. aureus cells at the site of infection becomes high, surface protein expression is reduced and exoprotein expression increases. The temporal regulation of surface proteins and exoproteins can be recapitulated in laboratory culture growth models, where early log phase growth represents an early infection and stationary phase represents late infection. Using this model system and classical genetics, two major pleiotropically-acting regulatory loci that govern temporal expression of surface proteins and exoproteins have been identified: agr, for accessory gene regulator (Recsei et al., 1986; Morfeldt et al., 1988; Peng et al., 1988) and sar, for staphylococcal accessory gene regulator (Cheung et al., 1992; Cheung and Projan, 1994). Mutations in these loci result in aberrant regulation of most virulence factors (e.g., lipase, coagulase, xcex1-toxin, adhesins, etc), which is reflected in diminished virulence in animal models of staphylococcal disease (Projan and Novick, 1997).
A scheme depicting the agr locus and its encoded proteins is shown in FIG. 1. Divergent promoters (P2 and P3), separated by approximately 180 bp, are responsible for transcription of the agrBDCA operon and RNAIII/hld operon (Morfeldt et al., 1996). The four Agr proteins combine to make a quorum-sensing system that is homologous to many two-component signal transduction systems found in prokaryotic organisms (Ji et al., 1997). AgrB is a cell membrane-bound transporter/processor of the AgrD peptide. AgrD is a 46 amino acid peptide that is cleaved to an octapeptide pheromone, exported by AgrB, and specifically recognized by (Ji et al., 1997) the AgrC membrane-bound receptor. The AgrD octapeptide pheromone allows an S. aureus cell to signal its presence to other cells in the growing colony. As the colony grows, the concentration of pheromone (Agr D) increases and reaches a particular level. AgrC, also an integral membrane protein, is activated by pheromone binding. AgrC is thought to be a kinase that acts on AgrA by initiating a signal transduction pathway that is believed to include AgrA. Whereas the exact mechanism of AgrA action is unknown, it is important for up-regulation of virulence gene expression and is thought to activate expression of the agr operon (RNAII) and the divergently expressed RNAIII. It is clear, however, from the work of Arvidson""s group (Morfeldt et al., 1996) that AgrA does not bind DNA either in the presence or absence of SarA. Mutations in any of the agr open reading frames (ORFs A, B, C, D) eliminate the up-regulation of RNAII and RNAIII expression (Novick et al., 1995). Additionally, agrA mutants have dramatically reduced virulence in animal models of staphylococcal arthritis, osteomyelitis, endocarditis and endopthalmitis (Abdelnour et al., 1993; Cheung et al., 1994a; Gillaspy et al., 1995; Booth et al., 1995). RNAIII is a regulatory RNA species, the function of which is not completely clear. However, there is evidence that RNAIII directly regulates expression of some S. aureus virulence genes by an anti-attenuation mechanism (Novick et al., 1993; Saravia-Otten et al., 1997).
In summary, when S. aureus attaches to a host tissue, a small amount of the AgrD pheromone is released. Early on, the concentration of the pheromone is too low to affect AgrC kinase activity. However, once the number of cells has risen, the local concentration of pheromone increases to a level whereby AgrC becomes activated. At this point the phenotypic switch is thrown and exoprotein expression dramatically increases.
Mutations in agr leads to decreased expression of exoprotein virulence factors and significantly reduced virulence in animal models of staphylococcal arthritis, endocarditis, osteomyelitis and endopthalmitis (Abdelnour et al., 1993; Cheung et al., 1994a; Gillaspy et al., 1995; Booth et al., 1995, respectively). Inhibition of the agr quorum-sensing/virulence gene activating system is a goal of the present invention. Since agr is activated by a transcriptional regulator, SarA, the present invention is directed to inhibiting this protein.
The second regulatory locus, sar, encodes a 14.4 kDa protein: SarA, also depicted in FIG. 1. Mutations in sar, like those in agr, lead to dramatically decreased virulence in animal models of staphylococcal disease (Cheung et al., 1994a). Interestingly, agrxe2x88x92, sarxe2x88x92 double mutants are less virulent than either of the single mutants in staphylococcal endocarditis, endophthalmitis and osteomyelitis (Cheung et al., 1994b; Booth et al., 1997; Gillaspy, et al., unpublished). Presumably, this phenotype is because SarA regulates expression of both transcripts in the agr locus (agr and RNAIII; Cheung et al., 1997) and SarA also regulates virulence factor genes that fall outside of agr control. For example, the cna gene, encoding the collagen adhesin, is not affected by mutations in the agr locus, but is under sar control (Gillaspy et al., 1998; Blevins et al., 1999). agr mutants do not have altered sar mRNA accumulation, whereas agr mRNA expression is dramatically affected by sar mutations (Cheung et al., 1997; Gillaspy and Smeltzer, unpublished). Specifically, there is a significant diminution of agr mRNA and nearly a complete loss of RNAIII in the sar strain ALC136 when compared to the wild type strain RN6390 (Cheung et al., 1997). The same observation has been made in clinical isolates in which the sar gene has been mutated (Gillaspy and Smeltzer, unpublished).
SarA present in crude extracts of S. aureus (Morfeldt et al., 1996) or recombinant SarA in E. coli extracts (Heinrichs et al., 1996) or purified, recombinant SarA (Chein and Cheung, 1998; U.S. Pat. No. 5,587,288; Rechtin et al, 1999,) have been shown to bind the DNA region of the agr promoters (this region also is referred to herein as the agr enhancer). AgrA has not been shown to bind DNA and was not present in the SarA-agr enhancer complexes (Morfeldt et al.,1996).
Arvidson and his colleagues recently reported that SarA is a DNA-binding regulatory protein and that its binding sites were located cis to the P2 and P3 promoters in the agr locus, a region that is referred to as the agr enhancer (Morfeldt et al., 1996). The agr enhancer has inverted repeats of a 7 bp sequence 5xe2x80x2-CTTAAGT-3xe2x80x2 (FIG. 2). Qualitative electrophoretic mobility shift assays (EMSA) were used to examine this region for regulatory proteins that may bind. Crude extracts of S. aureus wild type, sarxe2x88x92 and agrxe2x88x92 mutants were prepared. DNA fragments containing the left half of the region, right half of the region and the entire region were used in the EMSA tests. The migration of all of these fragments was retarded in native gels containing wild type and agrxe2x88x92 extracts, but not sarxe2x88x92 extracts. Furthermore, DNA affinity chromatography was used to purify the regulatory protein species and one protein with a MW of approximately 15 kDa was recovered. The amino acid sequence of the first 20 residues of that protein was determined and it matched that of the protein encoded by the sar gene. A simple model for SarA-mediated activation of genes in the agr locus would have SarA protein binding to a site that includes the heptad repeats and facilitating the binding of RNA polymerase to the adjacent promoters. However, complicating any simplified model is recent data from Rechtin et al. (1999) that unambiguously shows using high-affinity DNase I footprint analysis that the heptads are not the primary binding sites for SarA. Rather SarA protected three distinct, bipartite sites from DNase I digestion at extremely low protein concentrations, indicting very high affinity binding.
Production of the three distinct transcripts arising from the sar operon are regulated temporally (Bayer et al., 1996; Blevins et al., 1999). However, all three transcripts include the SarA ORF. Like agrA mutations, transposon insertions in the SarA ORF also eliminate induction of RNAII and RNAIII in late phase growth and result in reduced staphylococcal virulence in animal models of disease (Cheung et al., 1994 a and b; Booth et al., 1997). In seminal biochemical work in this area, SarA was shown to be a DNA-binding protein that is capable of binding DNA fragments containing cis regulatory elements for the promoters of both the agr operon (RNAII, P2 promoter) and the RNAIII operon (P3 promoter) (Morfeldt et al., 1996). Heptad repeats were identified upstream of both P2 and P3 promoters and were proposed to be SarA binding sites (see FIG. 2 and FIG. 3). A DNA fragment containing the RNAIII gene and 93 bp upstream of the transcription start site, including the heptad repeats, was sufficient for regulated expression of RNAII (see pEX085 in FIG. 3). Furthermore, removal of the distal half of the sequences upstream of the P3 promoter, including one heptad, eliminated appropriate expression of RNAIII (see pEX082 in FIG. 3). In addition, a synthetic DNA fragment including the repeats was bound by SarA in electrophoretic mobility shift assays (EMSA) in vitro using S. aureus extracts and was used successfully to purify SarA from extracts by DNA-affinity chromatography (Morfeldt et al., 1996).
In a more recent report, SarA, expressed as a GST-fusion protein in E. coli and purified, was observed to have relatively low affinity for DNA fragments containing the heptad repeats (Chien and Cheung, 1998). Furthermore, DNase I footprinting revealed a primary binding site for SarA in the inter-promoter region (see FIG. 3) that did not include the heptad repeats in the fragment cis to the P3 promoter shown to be sufficient for appropriate expression of RNAIII by Morfeldt et al. (1996). These two published reports of SarA interactions with the agr region have inconsistent conclusions regarding the binding sites for SarA in the agr locus (Morfeldt et al., 1996; Chien and Cheung, 1998). In the earlier report, approximately 60 bp upstream of the P3 promoter was shown to be sufficient for appropriate, regulated expression of RNAIII expression in S. aureus (Morfeldt et al., 1996). In addition, Morfeldt et al. (1996) proposed that SarA likely interacts with the regulatory regions containing the 7 bp repeats immediately upstream of the P3 and P2 promoters (see FIG. 2). However, in the latter report a DNase I footprint in the inter-promoter region using a recombinant fusion of GST-SarA was observed that had no overlap with the regulatory region described in the early report (Chien and Cheung, 1998).
The results of Chien and Cheung (1998) reporting an in vitro study of SarA-agr interactions may not be indicative of true SarA-agr interactions because of the nature of the protein used for the study. A GST-SarA fusion was used for most of the work and it appears that this construct most likely yielded an inactive SarA protein for two reasons: 1) After calculations of the concentrations of protein used in the EMSA and DNase I experiments reported in Chien and Cheung (1998), it became clear that micromolar amounts of SarA were required to achieve mobility shifts or footprints, respectively. This is a very high concentration of a DNA-binding protein. In the present invention, picomolar concentrations resulted in similar shifts in EMSA or footprints in DNase I protection experiments. Thus, the SarA protein used in the present invention appears to be several orders of magnitude more active than the protein used by Chien and Cheung. 2) Most importantly, to examine the monomer-dimer equilibrium for SarA, fluorescence anisotropy was used (Fernando and Royer, 1992; Maleki et al., 1997). For this work a fluorescent dye is coupled to the protein, typically the amino terminus. Fluoroscein, fluorescein with a six-carbon spacer and dansyl, independently, were coupled to the amino terminus of SarA, and in each case, a rapid inactivation of SarA was found as a result of unfolding (Hurlburt, unpublished). These dyes have been used in anisotropy experiments with several proteins previously without any problems (Maleki et al., 1997; Hurlburt unpublished). Thus, it was concluded that the amino terminus of SarA is intimately involved in SarA""s structural integrity and resultant activity. The determined low-resolution structure of SarA in the present invention shows that the amino terminus is proximal to the most likely DNA-binding domain of the protein. The SarA-GST fusion used by the Cheung group was to the amino terminus and it is strongly suspected that the low activity of the protein used in those studies was a result of the fusion.
In recent work, the quorum sensing mechanism encoded by the agr operon was inhibited and mice became protected from infection by S. aureus (Balaban et. al., 1998). This group approached the problem by targeting the quorum sensing signal molecule and its receptor. They looked at inhibitors of agr activation; i.e., the octapeptide pheromone and/or the 36 kDa protein. However, Richard Novick""s group reported that this kind of approach has a serious limitation (Ji et al., 1997). Namely, strains of S. aureus can be grouped based on whether they activate or inhibit the quorum sensing system encoded by agr. In other words, all strains autologously activate their own agr expression, but RN6390 inhibited agr expression in strains SA502, RN7843, RN8462 and RN8463, and vice versa in a process they termed bacterial interference. This likely is due to co-evolution of the signal molecule and its receptor. Apparently, the signal molecule from one strain inhibits the receptor of another, non-compatible strain. Thus, the Balaban approach may have limited efficacy against some strains of S. aureus. 
The present invention approaches the treatment of staphylococcal virulence and infection differently than previous publications by the inhibiting the activation of agr gene expression by inhibiting SarA function resulting in the inhibition of the expression of staphylococcal virulence factors. The present method of treatment provides a way to attenuate staphylococcal virulence which is believed to be more widely applicable than the Balaban inhibitor. This is so because SarA and its target DNA sequences cis to agr do not suffer as much strain variability as the Balaban inhibitor. Moreover, since the agr locus also includes RNAIII, a known regulator of virulence gene translation, inhibiting agr gene expression will have a more profound effect than inhibition of the quorum sensing system alone as disclosed by Balaban.
The present invention provides a novel method of treating staphylococcal diseases by interfering with the production of virulence factors, which in turn, prevents the Staphylococcus species from becoming a potent pathogen. The present invention is directed to designing, synthesizing and identifying potent inhibitors of SarA function and using these inhibitors to treat staphylococcal infections.
The present invention is directed to inhibitors of staphylococcal SarA protein function involved in the expression of staphylococcal virulence factors and the use of these inhibitors to treat and prevent staphylococcal infections in subjects. Particularly, the inhibitors act to interfere with the binding of the SarA protein to its binding site(s). The selection of specific inhibitors of the SarA protein is made possible as a result of the identification of the binding sites of SarA protein on at least a portion of the agr (accessory gene regulator) gene, a gene that like the sar (staphylococcal accessory regulator) gene, plays a role in the virulence of staphylococci species.
The present invention further is directed to a method of identifying inhibitors of SarA function involved in the expression of staphylococcal virulence genes comprising a) contacting a candidate inhibitor with at least one SarA binding site of the agr locus in solution to allow the binding reaction to equilibrate for a sufficient period of time; and b) assessing the binding of said candidate inhibitor to the SarA binding site of the agr locus.
The present invention further is directed to a method of identifying inhibitors of SarA function involved in the expression of staphylococcal virulence genes comprising a) contacting a candidate inhibitor with SarA in solution to allow the candidate inhibitor to affect the ability of SarA to bind to at least one SarA binding site of the agr locus; b) contacting said solution of step a) with at least one SarA binding site of the agr locus either simultaneously with the contact of said inhibitor and the SarA or subsequently to the contacting of the inhibitor and the SarA; and c) assessing the inhibition of the candidate inhibitor on the SarA binding to the SarA binding site of the agr locus.